Strong Cation Exchange Fractionation Research Articles

An Optimized Miniaturized Filter-Aided Sample Preparation Method for Sensitive Cross-Linking Mass Spectrometry Analysis of Microscale Samples.

Cross-linking mass spectrometry (XL-MS) is a powerful tool for elucidating protein structures and protein-protein interactions (PPIs) at the global scale. However, sensitive XL-MS analysis of mass-limited samples remains challenging, due to serious sample loss during sample preparation of the low-abundance cross-linked peptides. Herein, an optimized miniaturized filter-aided sample preparation (O-MICROFASP) method was presented for sensitive XL-MS analysis of microscale samples. By systematically investigating and optimizing crucial experimental factors, this approach dramatically improves the XL identification of low and submicrogram samples. Compared with the conventional FASP method, more than 7.4 times cross-linked peptides were identified from single-shot analysis of 1 μg DSS cross-linked HeLa cell lysates (440 vs 59). The number of cross-linked peptides identified from 0.5 μg HeLa cell lysates was increased by 58% when further reducing the surface area of the filter to 0.058 mm2 in the microreactor. To deepen the identification coverage of XL-proteome, five different types of cross-linkers were used and each μg of cross-linked HeLa cell lysates was processed by O-MICROFASP integrated with tip-based strong cation exchange (SCX) fractionation. Up to 2741 unique cross-linked peptides were identified from the 5 μg HeLa cell lysates, representing 2579 unique K-K linkages on 1092 proteins. About 96% of intraprotein cross-links were within the maximal distance restraints of 26 Å, and 75% of the identified PPIs reported by the STRING database were with high confidence (scores ≥0.9), confirming the high validity of the identified cross-links for protein structural mapping and PPI analysis. This study demonstrates that O-MICROFASP is a universal and efficient method for proteome-wide XL-MS analysis of microscale samples with high sensitivity and reliability.

Diagonal Chromatography for the Isolation of O-Linked β-N-acetyl-D-Glucosaminylated (O-GlcNAcylated) Peptides in HeLa Cell Extracts

Protein O-linked-N-acetylglucosaminylation (O-GlcNAcylation) is a dynamic post-translational modification process that plays an essential role in biological activities. Growing evidence indicates that aberration of O-GlcNAcylation is associated with various diseases, e.g. diabetes, neurological diseases, and cancers. However, the mechanistic studies of O-GlcNAcylation are lagging behind other post-translational modifications due to its extremely low abundance, limited analytical tools, and specificity. Herein, diagonal strong cation exchange chromatography was applied to enrich the O-GlcNAc glycosylated peptides prior to mass spectrometric analysis by liquid chromatography/ion trap tandem mass spectrometry (LC–MS/MS). In this strategy, the O-GlcNAcylated peptides were first enzymatically labeled with an azide-modified galactosamine (GalNAz) and fractionated by strong cation exchange (SCX) chromatography. Tris(carboxyethyl)phosphine (TCEP) reduces the azido group in GalNAz-modified peptides to a primary amine group. TCEP-induced reduction of GalNAz-modified peptides was separated from unmodified peptides by diagonal SCX. By reversed-phase LC–MS/MS analysis of secondary SCX fractions, O-GlcNAcylated peptides were isolated and identified from the mixtures of O-GlcNAc-modified and unmodified peptides in HeLa cell extract. A total of 250 O-GlcNAcylation sites on 215 proteins were identified. Therefore, this novel method could be a potential tool for the isolation and site analysis of O-GlcNAc-modified peptides.

Identification of antioxidant peptides derived from tropical jackfruit seed and investigation of the stability profiles

Jackfruit is a sweet tropical fruit with very pleasant aroma, and the ripe seeds are edible. In this study, jackfruit seed proteins were isolated and subjected to trypsin digestion. The resultant protein hydrolysate was then subjected to antioxidant assay-guided purification, using centrifugal filtration, C18 reverse-phase and strong cation exchange (SCX) fractionations. The purified SCX fraction was further analyzed by de novo peptide sequencing, and two peptide sequences were identified and synthesized. Peptide JFS-2 (VGPWQK) was detected with antioxidant potential, with EC50 value comparable to that of commercial GSH antioxidant peptide. Additionally, the identified peptides were tested with protein protection potential, in an albumin protein denaturation inhibitory assay. Concurrently, we also investigated the pH, temperature, and gastrointestinal-digestion stability profiles for the identified peptide. With further research efforts, the identified peptides could potentially be developed into preservative agent for protein-rich food systems or as health-promoting diet supplements.

Identification of a potent Angiotensin-I converting enzyme inhibitory peptide from Black cumin seed hydrolysate using orthogonal bioassay-guided fractionations coupled with in silico screening

Black cumin (Nigella sativa) seed protein (BCSP) was individually hydrolyzed with pepsin, trypsin, and α-chymotrypsin. After ultrafiltration, the α-chymotrypsin hydrolysate (< 3 kDa) exhibited the highest ACE inhibitory (ACEI) activity with an IC50 value of 34.4 ± 1.5 μg/mL. This hydrolysate was orthogonally fractionalized using reversed-phase high-performance liquid chromatography (RP-HPLC) and strong cation exchange (SCX) chromatography, and the most active RP-HPLC and SCX fractions (F7 and H4, respectively) were individually screened out by ACEI assay. These two fractions were analyzed with liquid chromatography-tandem mass spectrometry (LC–MS/MS) followed by automated de novo peptide sequencing, and totally 43 ACEI candidate peptides were identified. Three overlapping peptides (VTPVGVPKW, VVTPVGVPKW, and LVLTL) were simultaneously contained in both fractions, and VTPVGVPKW (VW-9) was speculated as to the most potent ACEI peptide based on the in silico analysis. Synthetic VW-9 was used to confirm the identity, and a remarkable IC50 value of VW-9 (1.8 ± 0.09 μM) was determined. Preincubation and inhibition mechanism studies indicated that VW-9 was a true inhibitor as well as a non-competitive inhibitor on ACE, which was further illustrated with the molecular docking simulation. Our study revealed that the application of VW-9 to antihypertensive products is promising.

Preparation of Mitochondria from Ovarian Cancer Tissues and Control Ovarian Tissues for Quantitative Proteomics Analysis.

Ovarian cancer is a common gynecologic cancer with high mortality but unclear molecular mechanism. Most ovarian cancers are diagnosed in the advanced stage, which seriously hampers therapy. Mitochondrial changes are a hallmark of human ovarian cancers, and mitochondria are the centers of energy metabolism, cell signaling, and oxidative stress. In-depth insights into the changes of the mitochondrial proteome in ovarian cancers compared to control ovarian tissue will benefit in-depth understanding of the molecular mechanisms of ovarian cancer, and the discovery of effective and reliable biomarkers and therapeutic targets. An effective mitochondrial preparation method coupled with an isobaric tag for relative and absolute quantification (iTRAQ) quantitative proteomics are presented here to analyze human ovarian cancer and control mitochondrial proteomes, including differential-speed centrifugation, density gradient centrifugation, quality assessment of mitochondrial samples, protein digestion with trypsin, iTRAQ labeling, strong cation exchange fractionation (SCX), liquid chromatography (LC), tandem mass spectrometry (MS/MS), database analysis, and quantitative analysis of mitochondrial proteins. Many proteins have been successfully identified to maximize the coverage of the human ovarian cancer mitochondrial proteome and to achieve the differentially expressed mitochondrial protein profile in human ovarian cancers.

Discovery and Study of Novel Antihypertensive Peptides Derived from Cassia obtusifolia Seeds.

Antihypertensive peptides were screened from thermolysin hydrolysate of Cassia obtusifolia seeds (Jue Ming Zi) using two independent bioassay-guided fractionations, reversed-phase high-performance liquid chromatography (RP-HPLC), and strong cation-exchange (SCX) liquid chromatography coupled with angiotensin I-converting enzyme (ACE) inhibitory assay. The identical peptide in the most active RP-HPLC and SCX fractions was simultaneously de novo sequenced as FHAPWK with high-resolution mass spectrometry. FHAPWK (IC50 = 16.83 ± 0.90 μM) was further identified as a competitive inhibitor and a true inhibitor on ACE by a Lineweaver-Burk plot and preincubation experiment, respectively. The molecular docking simulation indicated that FHAPWK could interact with several key residues of the ACE active site, which is consistent with the result of the inhibitory kinetics study. Moreover, its antihypertensive effect was demonstrated using the animal model of spontaneously hypertensive rats. It is concluded that FHAPWK is the first reported antihypertensive peptide derived from thermolysin hydrolysate ofC. obtusifolia seeds.

Proteome Analysis of Human Neutrophil Granulocytes From Patients With Monogenic Disease Using Data-independent Acquisition

Neutrophil granulocytes are critical mediators of innate immunity and tissue regeneration. Rare diseases of neutrophil granulocytes may affect their differentiation and/or functions. However, there are very few validated diagnostic tests assessing the functions of neutrophil granulocytes in these diseases. Here, we set out to probe omics analysis as a novel diagnostic platform for patients with defective differentiation and function of neutrophil granulocytes. We analyzed highly purified neutrophil granulocytes from 68 healthy individuals and 16 patients with rare monogenic diseases. Cells were isolated from fresh venous blood (purity >99%) and used to create a spectral library covering almost 8000 proteins using strong cation exchange fractionation. Patient neutrophil samples were then analyzed by data-independent acquisition proteomics, quantifying 4154 proteins in each sample. Neutrophils with mutations in the neutrophil elastase gene ELANE showed large proteome changes that suggest these mutations may affect maturation of neutrophil granulocytes and initiate misfolded protein response and cellular stress mechanisms. In contrast, only few proteins changed in patients with leukocyte adhesion deficiency (LAD) and chronic granulomatous disease (CGD). Strikingly, neutrophil transcriptome analysis showed no correlation with its proteome. In case of two patients with undetermined genetic causes, proteome analysis guided the targeted genetic diagnostics and uncovered the underlying genomic mutations. Data-independent acquisition proteomics may help to define novel pathomechanisms in neutrophil diseases and provide a clinically useful diagnostic dimension.

Accurate Quantitative Proteomic Analyses Using Metabolic Labeling and High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS).

Stable isotope labeling by amino acids in cell culture (SILAC) is routinely used to profile changes in protein and peptide abundance across different experimental paradigms. As with other quantitative proteomic approaches, the detection of peptide isotopomers can be limited by the presence of interference ions that ultimately affect the quality of quantitative measurements. Here, we evaluate high field asymmetric waveform ion mobility spectrometry (FAIMS) to improve the accuracy and dynamic range of quantitative proteomic analyses using SILAC. We compared quantitative measurements for tryptic digests of isotopically labeled protein extracts mixed in different ratios using LC-MS/MS with and without FAIMS. To further reduce sample complexity, we also examined the improvement in quantitative measurements when combining strong cation exchange (SCX) fractionation prior to LC-MS/MS analyses. Using the same amount of sample consumed, analyses performed using FAIMS provided more than 30% and 200% increase in the number of quantifiable peptides compared to LC-MS/MS performed with and without SCX fractionation, respectively. Furthermore, FAIMS reduced the occurrence of interfering isobaric ions and improved the accuracy of quantitative measurements. We leveraged the application of FAIMS in phosphoproteomic analyses to profile dynamic changes in protein phosphorylation in HEK293 cells subjected to heat shock for periods up to 20 min. In addition to the enhanced phosphoproteomic coverage, FAIMS also provided the ability to separate phosphopeptide isomers that often coelute and can be misassigned in conventional LC-MS/MS experiments.

SILProNAQ: A Convenient Approach for Proteome-Wide Analysis of Protein N-Termini and N-Terminal Acetylation Quantitation.

Protein N-terminal modifications have recently been involved in overall proteostasis through their impact on cell fate and protein life time. This explains the development of new approaches to characterize more precisely the N-terminal end of mature proteins. Although few approaches are available to perform N-terminal enrichment based on positive or negative discriminations, these methods are usually restricted to the enrichment in N-terminal peptides and their characterization by mass spectrometry. Recent investigation highlights both (1) the knowledge of the N-terminal acetylation status of most cytosolic proteins and (2) post-translational addition of this modification on the N-terminus of nuclear coded chloroplast proteins imported in the plastid and after the cleavage of the transit peptide. The workflow involves stable isotope labeling to assess N-acetylation rates followed by Strong Cation eXchange (SCX ) fractionation of the samples to provide protein N-terminal enriched fractions. Combined with mass spectrometry analyses, the technology finally requires extensive data processing. This last step aims first at discriminating the most relevant mature N-termini from the characterized peptides, next at determining its experimental position and then at calculating the N-terminal acetylation yield. Stable-Isotope Protein N-terminal Acetylation Quantification (SILProNAQ) is a complete workflow combining wet-lab techniques together with dry-lab processing to determine the N-terminal acetylation yield of mature proteins for a clearly defined localization.

Protein Isolation from Plasma Membrane, Digestion and Processing for Strong Cation Exchange Fractionation.

Plasma membrane (PM) proteins play crucial roles in diverse biological processes. But their low abundance, alkalinity and hydrophobicity make their isolation a difficult task. This protocol describes an efficient method for PM proteins isolation, digestion and fractionation so that they can be well prepared for mass spectrometry analysis.

Identification of Carboxypeptidase Substrates by C-Terminal COFRADIC.

We here present a detailed procedure for studying protein C-termini and their posttranslational modifications by C-terminal COFRADIC. In fact, this procedure can enrich for both C-terminal and N-terminal peptides through a combination of a strong cation exchange fractionation step at low pH, which removes the majority of nonterminal peptides in whole-proteome digests, while the actual COFRADIC step segregates C-terminal peptides from N-terminal peptides. When used in a differential mode, C-terminal COFRADIC allows for the identification of neo-C-termini generated by the action of proteases, which in turn leads to the identification of protease substrates. More specifically, this technology can be applied to determine the natural substrate repertoire of carboxypeptidases on a proteome-wide scale.

Screening of angiotensin-I converting enzyme inhibitory peptides derived from soft-shelled turtle yolk using two orthogonal bioassay-guided fractionations

Two orthogonal bioassay-guided fractionations were simultaneously applied to screen angiotensin-I converting enzyme inhibitory (ACEI) peptides from soft-shelled turtle yolk (SSTY) hydrolysate. Peptides derived from thermolysin digest of SSTY were fractionated separately using reversed phased (RP) and strong cation exchange (SCX) chromatography, then the inhibitory activities of resulting fractions were examined using ACEI assay. The peptides in the most active RP and SCX fractions were characterized by liquid chromatography-tandem mass spectrometry (LC–MS/MS) to give 11 and 35 non-redundant peptides, respectively. AKLPSW, simultaneously found in the active RP and SCX fractions, was identified as the most effective ACEI peptide. This identity of AKLPSW was further confirmed using synthetic peptide and its IC50 value was determined as 15.3±0.9μM. The kinetic study revealed that AKLPSW is a noncompetitive inhibitor and its interaction towards ACE was proposed using molecular docking. Furthermore, the antihypertensive effect of AKLPSW was demonstrated using spontaneously hypertensive rats.

Identification of Acetaminophen Adducts of Rat Liver Microsomal Proteins using 2D-LC-MS/MS.

Xenobiotic metabolism in the liver can give rise to reactive metabolites that covalently bind to proteins, and determining which proteins are targeted is important in drug discovery and molecular toxicology. However, there are difficulties in the analysis of these modified proteins in complex biological matrices due to their low abundance. In this study, an analytical approach was developed to systematically identify target proteins of acetaminophen (APAP) in rat liver microsomes (RLM) using two-dimensional chromatography and high-resolution tandem mass spectrometry. In vitro microsomal incubations, with and without APAP, were digested and subjected to strong cation exchange (SCX) fractionation prior to reverse-phase UHPLC-MS/MS. Four data processing strategies were combined into an efficient label-free workflow meant to eliminate potential false positives, using peptide spectral matching, statistical differential analysis, product ion screening, and a custom-built delta-mass filtering tool to pinpoint potential modified peptides. This study revealed four proteins, involved in important cellular processes, to be covalently modified by APAP. Data are available via ProteomeXchange with identifier PXD002590.

Online coupling of hydrophilic interaction/strong cation exchange/reversed-phase liquid chromatography with porous graphitic carbon liquid chromatography for simultaneous proteomics and N-glycomics analysis

In this study we developed a fully automated three-dimensional (3D) liquid chromatography methodology—comprising hydrophilic interaction separation as the first dimension, strong cation exchange fractionation as the second dimension, and low-pH reversed-phase (RP) separation as the third dimension—in conjunction downstream with additional complementary porous graphitic carbon separation, to capture non-retained hydrophilic analytes, for both shotgun proteomics and N-glycomics analyses. The performance of the 3D system alone was benchmarked through the analysis of the total lysate of Saccharomyces cerevisiae, leading to improved hydrophilic peptide coverage, from which we identified 19% and 24% more proteins and peptides, respectively, relative to those identified from a two-dimensional hydrophilic interaction liquid chromatography and low-pH RP chromatography (HILIC–RP) system over the same mass spectrometric acquisition time; consequently, the 3D platform also provided enhanced proteome and protein coverage. When we applied the integrated technology to analyses of the total lysate of primary cerebellar granule neurons, we characterized a total of 2201 proteins and 16,937 unique peptides for this primary cell line, providing one of its most comprehensive datasets. Our new integrated technology also exhibited excellent performance in the first N-glycomics analysis of cynomolgus monkey plasma; we successfully identified 122 proposed N-glycans and 135 N-glycosylation sites from 122 N-glycoproteins, and confirmed the presence of 38 N-glycolylneuraminic acid-containing N-glycans, a rare occurrence in human plasma, through tandem mass spectrometry for the first time.

Workflow for large-scale analysis of melanoma tissue samples

The aim of the present study was to create an optimal workflow for analysing a large cohort of malignant melanoma tissue samples. Samples were lysed with urea and enzymatically digested with trypsin or trypsin/Lys C. Buffer exchange or dilution was used to reduce urea concentration prior to digestion. The tissue digests were analysed directly or following strong cation exchange (SCX) fractionation by nano LC–MS/MS. The approach which resulted in the largest number of protein IDs involved a buffer exchange step before enzymatic digestion with trypsin and chromatographic separation in 120min gradient followed by SCX–RP separation of peptides.

Comparison of fractionation strategies for offline two-dimensional liquid chromatography tandem mass spectrometry analysis of proteins from mouse adipose tissue

In the frame of protein identification from mouse adipose tissue, two strategies were compared for the offline elution of peptides from a strong cation exchange (SCX) column in two-dimensional liquid chromatography tandem mass spectrometry (2D–LC–MS/MS) analyses. First, the salt gradient (using K+ as displacing agent) was evaluated from 25 to 500mM KCl. Then, a less investigated elution mode using a pH gradient (using citric acid and ammonium hydroxide) was carried out from pH 2.5 to 9.0. Equal amounts of peptide digest derived from mouse adipose tissue were loaded onto the SCX column and fractionated according to the two approaches. A total of 15 fractions were collected in two independent experiments for each SCX elution strategy. Then, each fraction was analyzed on a nanoLC–MS/MS platform equipped with a column-switching unit for desalting and enrichment. No substantial differences in peptide quality characteristics (molecular weight, isoelectric point, or GRAVY [grand average of hydropathicity] index distributions) were observed between the two datasets. The pH gradient approach was found to be superior, with 27.5% more unique peptide identifications and 10% more distinct protein identifications compared with the salt-based elution method. In conclusion, our data imply that the pH gradient SCX fractionation is more desirable for proteomics analysis of entire adipose tissue.

SWATH analysis of the synaptic proteome in Alzheimer's disease

Brain tissue from Alzheimer's disease patients exhibits synaptic degeneration in selected regions. Synaptic dysfunction occurs early in the disease and is a primary pathological target for treatment. The molecular mechanisms underlying this degeneration remain unknown. Quantifying the synaptic proteome in autopsy brain and comparing tissue from Alzheimer's disease cases and subjects with normal aging are critical to understanding the molecular mechanisms associated with Alzheimer pathology. We isolated synaptosomes from hippocampus and motor cortex so as to reduce sample complexity relative to whole-tissue homogenates. Synaptosomal extracts were subjected to strong cation exchange (SCX) fractionation to further partition sample complexity; each fraction received SWATH-based information-dependent acquisition to generate a comprehensive peptide-ion library. The expression of synaptic proteins from AD hippocampus and motor cortex was then compared between groups. A total of 2077 unique proteins were identified at a critical local false discovery rate <5%. Thirty of these, including 17 novel proteins, exhibited significant expression differences between cases and controls; these proteins are involved in cellular functions including structural maintenance, signal transduction, autophagy, oxidative stress, and proteasome activity, or they have synaptic-vesicle related or energy-related functions. Differentially expressed proteins were subjected to pathway analysis to identify protein–protein interactions. This revealed that the most perturbed molecular and cellular functions were cellular assembly and organization. Core analysis revealed RhoA signaling to be the top canonical pathway. Network analysis showed that differentially expressed proteins were related to cellular assembly and organization, and cellular function and maintenance. This is the first study to combine SCX fractionation with SWATH analysis. SWATH is a promising new technique that can greatly enhance protein identification in any proteome, and has many other benefits; however, there are limitations yet to be resolved.

Proteomic analysis of ligamentum flavum from patients with lumbar spinal stenosis.

Lumbar spinal stenosis (LSS) is a syndromic degenerative spinal disease and is characterized by spinal canal narrowing with subsequent neural compression causing gait disturbances. Although LSS is a major age-related musculoskeletal disease that causes large decreases in the daily living activities of the elderly, its molecular pathology has not been investigated using proteomics. Thus, we used several proteomic technologies to analyze the ligamentum flavum (LF) of individuals with LSS. Using comprehensive proteomics with strong cation exchange fractionation, we detected 1288 proteins in these LF samples. A GO analysis of the comprehensive proteome revealed that more than 30% of the identified proteins were extracellular. Next, we used 2D image converted analysis of LC/MS to compare LF obtained from individuals with LSS to that obtained from individuals with disc herniation (nondegenerative control). We detected 64781 MS peaks and identified 1675 differentially expressed peptides derived from 286 proteins. We verified four differentially expressed proteins (fibronectin, serine protease HTRA1, tenascin, and asporin) by quantitative proteomics using SRM/MRM. The present proteomic study is the first to identify proteins from degenerated and hypertrophied LF in LSS, which will help in studying LSS.

Quantitative proteomic analysis of serum proteins from oral cancer patients: comparison of two analytical methods.

Serum proteomic analysis can be a valuable approach for the discovery of protein biomarkers for early detection or monitoring of a disease. In this study, two analytical methods were compared for quantification of serum proteins in patients with oral cancer. In the first approach, we quantified serum proteins between oral squamous cell carcinoma (OSCC) and healthy control subjects by performing in-solution digestion of serum proteins, isobaric tags for relative and absolute quantification (iTRAQ) labeling of the resulting peptides, strong cation exchange (SCX) fractionation of labeled peptides and finally capillary liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis of the peptides. In the second approach, we first separated serum proteins with SDS-PAGE. The gel-separated proteins were then digested with trypsin and the resulting peptides were labeled with iTRAQ and analyzed with LC-MS/MS for protein quantification. A total of 319 serum proteins were quantified with the first proteomic approach whereas a total of 281 proteins were quantified by the second proteomic approach. Most of the proteins were identified and quantified by both approaches, suggesting that these methods are similarly effective for serum proteome analysis. This study provides compelling evidence that quantitative serum proteomic analysis of OSCC is a valuable approach for identifying differentially expressed proteins in cancer patients’ circulation systems that may be used as potential biomarkers for disease detection. Further validation in large oral cancer patient populations may lead to a simple and low invasive clinical tool for OSCC diagnosis or monitoring.

Use of singular value decomposition analysis to differentiate phosphorylated precursors in strong cation exchange fractions.

We studied the use of peak deviations (PDs) for application in phosphoproteomics. Due to the differences in the mass defects, the PDs of samples containing mixtures of phosphorylated and nonphosphorylated peptides show bimodal distributions. The ratios of peak heights accurately predict the phosphoproteome content of a sample. In this work, we apply a signal-processing tool, singular value decomposition, to reveal characteristic features of the phosphorylated, nonphosphorylated, and mixed samples. We show that a simple application of singular value decomposition to the PD matrix (i) detects transitions from mostly phosphorylated samples to mostly nonphosphorylated samples, (ii) reveals modes of low-abundance species in the presence of the high-abundance species (e.g., phosphorylated peptides), and (iii) simplifies the interpretation of the clustering of a covariance matrix obtained from PDs. As the eigenfunctions of the inner-product of the data matrix (made from the PDs) are Hermite functions, we observe a change of sign in the transition from samples enriched in phosphorylated peptides to samples containing fewer phosphorylated peptides. The ordering of the singular values of the data matrix points in the direction of changes to the phosphorylation content. No peptide identifications from a database were used for this study.